Auto-focusing method and device

Abstract

An auto-focusing method and device are presented for determining an in-focus position of a sample supported on a substrate plate made of a material transparent with respect to incident electromagnetic radiation. The method utilizes an optical system capable of directing incident electromagnetic radiation towards the sample and collecting reflections of the incident electromagnetic radiation that are to be detected. A focal plane of an objective lens arrangement is located at a predetermined distance from a surface of the substrate, which is opposite to the sample-supporting surface of the substrate. A continuous displacement of the focal plane relative to the substrate along the optical axis of the objective lens arrangement is provided, while concurrently directing the incident radiation towards the sample through the objective lens arrangement to thereby focus the incident radiation to a location at the focal plane of the objective lens arrangement. Reflected components of the electromagnetic radiation to a location objective lens arrangement are continuously detected. The detected reflected components are characterized by a first intensity peak corresponding to an in-focus position of said opposite surface of the substrate, and a second intensity peak spaced in time from the first intensity peak and corresponding to an in-focus position of said sample-supporting surface of the substrate. This technique enables imaging of the sample when in the in-focus position of the sample-supporting surface of the substrate.

Description

FIELD OF THE INVENTION [0001] This invention is generally in the field of optical measurement / inspection techniques and relates to an auto-focus method and device, and an imaging system utilizing the same for use with optical microscopy. The invention is particularly useful for the microscope inspection of fluorescently labeled biological specimens on transparent slides. BACKGROUND OF THE INVENTION [0002] Auto focusing is an essential feature in many automated inspection fields such as the chip industry, biomedical research, data reading / recording in optical information carriers, etc. [0003] Auto-focusing techniques used in the inspection / measurement of such structures as semiconductor wafers typically utilize an optical system separated from an imaging optics, which projects a light pattern (e.g., spots, lines, etc.) on the wafer and optimizes the image of this pattern (by using intensity contrast or displacement). Such techniques are applicable only for long working distance micro...

AI Technical Summary

Benefits of technology

The present invention provides a method and device for automatically focusing on a sample supported on a substrate plate made of a material transparent to incident radiation. This is achieved by detecting the in-focus position of the sample by continuously varying the relative distance between the objective lens arrangement and the sample while directing the incident radiation towards the sample. The technique uses a sweeping focus mode, which allows for a very fast approach to focus but no mechanical overshoot when reaching the in-focus position of the substrate's surface. The device includes a light source, focusing optics, and a light directing assembly. The invention enables quick and accurate auto-focal positioning of samples, particularly biological samples with fluorescent labels, for imaging.

Problems solved by technology

These techniques require calibrations and adjustments to match auto-focusing and imaging optical systems, and are hard to implement, especially when the imaging optics has variable magnification.

Additionally, these techniques are not preserving intensities, and therefore are not applicable for quantitative fluorescence microscopy applications.

Such accuracy is far beyond the tolerance of the bottom surfaces of multi-well dishes, and even typical high-performance XYZ stages cannot scan distances of about 10 cm in the XY plane while keeping the Z coordinate within the above tolerances.

However, this approach has several inherent problems: 1.

The process is slow.

Yet, there is no scheme that will map the distance from the focus into any evaluation parameter with smooth dependence displaying a single minimum over three orders of magnitude range of defocusing.

Glass slides or plastic well bottoms are a fraction of a millimeter thick and may often cause auto-focus devices based on maximal sharpness to focus at the wrong side of the sample substrate.

However, image-based focusing is not applicable to fluorescent microscopy, where repeated imaging may cause bleach and phototoxicity.

Focusing methods that evaluate fluorescence are therefore not applicable for such screens.

Images